Evaluating State Dependence and Subtype Selectivity of Calcium Channel Modulators in Automated Electrophysiology Assays.
ABSTRACT Abstract Voltage-gated Ca(2+) channels play essential roles in control of neurosecretion and muscle contraction. The pharmacological significance of Cav channels stem from their identification as the molecular targets of calcium blockers used in the treatment of cardiovascular diseases, such as hypertension, angina, and arrhythmia, and neurologic diseases, such as pain and seizure. It has been proposed that state-dependent Cav inhibitors, that is, those that preferentially bind to channels in open or inactivated states, may improve the therapeutic window over relatively state-independent Cav inhibitors. High-throughput fluorescent-based functional assays have been useful in screening chemical libraries to identify Cav inhibitors. However, hit confirmation, mechanism of action, and subtype selectivity are better suited to automated patch clamp assays that have sufficient capacity to handle the volume of compounds identified during screening, even of modest sized libraries (≤500,000 compounds), and the flexible voltage control that allows evaluation of state-dependent drug blocks. IonWorks™ Barracuda (IWB), the newest generation of IonWorks instruments, provides the opportunity to accelerate the Cav drug discovery studies in an automated patch clamp platform in 384-well format capable of medium throughput screening and profiling studies. We have validated hCav1.2, hCav2.1, hCav2.2, and hCav3.2 channels assays on the IWB platform (population patch clamp mode) and demonstrated that the biophysical characteristics of the channels (activation, inactivation, and steady-state inactivation) obtained with the IWB system are consistent with known subtype-specific characteristics. Using standard reference compounds (nifedipine, BAY K8644, verapamil, mibefradil, and pimozide), we demonstrated subtype-selective and state- and use-dependent characteristics of drug-channel interactions. Here we describe the design and validation of novel robust high-throughput Cav channel assays on the IWB platform. The assays can be used to screen focused compound libraries for state-dependent Cav channel antagonists, to prioritize compounds for potency or to counterscreen for Cav subtype selectivity.
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ABSTRACT: The normal electrophysiologic behavior of the heart is determined by the integrated activity of specific cardiac ionic currents. Mutations in genes encoding the molecular components of individual cardiac ion currents have been shown to result in multiple cardiac arrhythmia syndromes. Presently, 12 genes associated with inherited long QT syndrome (LQTS) have been identified, and the most common mutations are in the hKCNQ1 (LQT1, Jervell and Lange-Nielson syndrome), hKCNH2 (LQT2), and hSCN5A (LQT3, Brugada syndrome) genes. Several drugs have been withdrawn from the market or received black box labeling due to clinical cases of QT interval prolongation, ventricular arrhythmias, and sudden death. Other drugs have been denied regulatory approval owing to their potential for QT interval prolongation. Further, off-target activity of drugs on cardiac ion channels has been shown to be associated with increased mortality in patients with underlying cardiovascular diseases. Since clinical arrhythmia risk is a major cause for compound termination, preclinical profiling for off-target cardiac ion channel interactions early in the drug discovery process has become common practice in the pharmaceutical industry. In the present study, we report assay development for three cardiac ion channels (hKCNQ1/minK, hCa(v)1.2, and hNa(v)1.5) on the IonWorks Quattro™ system. We demonstrate that these assays can be used as reliable pharmacological profiling tools for cardiac ion channel inhibition to assess compounds for cardiac liability during drug discovery.Assay and Drug Development Technologies 12/2010; 8(6):766-80. · 2.08 Impact Factor
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ABSTRACT: Abstract Ca(V)2.2 (N-type) calcium channels are key regulators of neurotransmission. Evidence from knockout animals and localization studies suggest that Ca(V)2.2 channels play a critical role in nociceptive transmission. Additionally, ziconotide, a selective peptide inhibitor of Ca(V)2.2 channels, is clinically used to treat refractory pain. However, the use of ziconotide is limited by its low therapeutic index, which is believed, at least in part, to be a consequence of ziconotide inhibiting Ca(V)2.2 channels regardless of the channel state. Subsequent efforts have focused on the discovery of state-dependent inhibitors that preferentially bind to the inactivated state of Ca(V)2.2 channels in order to achieve an improved safety profile relative to ziconotide. Much less attention has been paid to understanding the binding kinetics of these state-dependent inhibitors. Here, we describe a novel electrophysiology-based assay on an automated patch platform designed to differentiate Ca(V)2.2 inhibitors based on their combined state dependence and kinetics. More specifically, this assay assesses inactivated state block, closed state block, and monitors the kinetics of recovery from block when channels move between states. Additionally, a use-dependent assay is described that uses a train of depolarizing pulses to drive channels to a similar level of inactivation for comparison. This use-dependent protocol also provides information on the kinetics of block development. Data are provided to show how these assays can be utilized to screen for kinetic diversity within and across chemical classes.Assay and Drug Development Technologies 03/2012; · 2.08 Impact Factor
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ABSTRACT: The automated electrophysiology platform IonWorks has facilitated the medium-throughput study of ion channel biology and pharmacology. Electrical and chemical access to the cell is by perforated patch, afforded by amphotericin. Permeation of the amphotericin pore is limited to monovalent cations. We describe here the use of the saponin escin as an alternative perforating agent. With respect to the number and robustness of seals formed across a variety of cell and ion channel types, the performance of escin is equal to that of amphotericin. Escin also permits the permeation of larger molecules through its pore. These include nucleotides, important intracellular modulators of ion channel activity that can be used to prevent ion channel rundown of, for instance, Ca(V)1.2. Furthermore, pharmacologic agents such as QX314 can also permeate and be used for mechanistic studies. Escin, in combination with IonWorks, increases the scope of ion channel screening and can facilitate the assay of previously difficult-to-assay targets.Journal of Biomolecular Screening 08/2012; · 2.01 Impact Factor